Certain biological molecules or "biomolecules", such as enzymes, antibodies and nucleic acids, possess reactive recognition properties for other molecular species ("substrates"), giving rise to specific complexation, reaction and product formation. Electrochemical biosensors are a class of sensors in which electrochemistry provides a means of signal transduction, either through detection of products of enzyme reactions or of the concentration of electroactive tags used in competitive binding or sandwich immunoassays. The signal may be due to a change in current or in potential on the electrode ("amperometric" or "potentiometric" detection, respectively). A particularly useful type of electrochemical biosensor is produced when the biomolecule is immobilized onto the electrode surface. Electrodes with immobilized enzymes are widely used in glucose monitoring for insulin maintenance, for example.
A common class of biomolecules used to prepare electrochemical biosensors are the so-called "oxidoreductase" enzymes, of which glucose oxidase (GOx) is an illustrative example. GOx catalyzes the reaction between glucose and O.sub.2 to produce gluconolactone and H.sub.2 O.sub.2. The reaction occurs via the intermediate flavin adenosine dinucleotide oxidation-reduction ("redox") system FAD/FADH.sub.2, which is incorporated inside the enzyme's protein envelope. Thus, the full reaction is: EQU glucose+GOx(FAD).fwdarw.gluconolactone+GOx(FADH.sub.2)GOx(FADH.sub.2)+O.sub .2 .fwdarw.FAD+H.sub.2 O.sub.2
If the electrode is maintained at a potential sufficiently positive to oxidize the H.sub.2 O.sub.2, the current due to H.sub.2 O.sub.2 oxidation provides a reading of glucose concentration: EQU H.sub.2 O.sub.2 .fwdarw.2H.sup.+ +O.sub.2 +2e.sup.-
In amperometric biosensors such as this it is required that the electrochemically active product, be it a freely diffusing product or an electron shuttle contacting the enzyme, efficiently transfer electrons to or from the electrode so that the electrical current can be measured by the external circuit. Enzymes can be immobilized into nonconductive polymer matrices coated onto the surface of electrodes. Such matrices can be made permeable to both substrate and product. However, response may be reduced by the necessity of diffusion of the product through the matrix to the electrode surface. To alleviate this problem, immobilization has been achieved in electrically conductive matrices by numerous means in prior art. For example, matrices composed of platinized, resin bound carbon or graphite particles have been disclosed in U.S. Pat. Nos. 4,970,145 and 5,160,418. Inclusion of Pt oxide in carbon or graphite enzyme electrodes to reduce their activity to alcohols is described in U.S. Pat. No. 5,231,028.
Electrically conductive polymers such as polypyrrole, polyaniline and polythiophene have been demonstrated as encapsulants for oxidoreductase enzymes, see for example M. Trojanowicz et al. (Sensors and Actuators B28, 191-199, 1995) and Hiller et al. Adv. Mater. 8:219-222, 1996) and references cited therein. These conductive or redox polymer matrices serve the dual function of trapping the enzyme and conducting electrons to or from the enzyme, electron transfer mediators or reaction products and the electrode surface. They are also sufficiently porous to provide free transport of electrolyte, substrate and products. Conductive polymer layers containing enzyme may be formed by electropolymerizing the monomer onto the electrode surface in the presence of the enzyme. They may also be formed by biasing a preformed polymer coating in the presence of the enzyme in solution at a positive potential, thus attracting the negatively charged enzyme electrostatically to the polymer matrix.
Conducting polymer encapsulants have several drawbacks that are well known for this class of materials, including poor long-term stability to water, oxygen and temperature, poor stability to products of enzyme reactions (e.g., H.sub.2 O.sub.2), lack of physical toughness, potential toxicity or instability when implanted into live organisms, poor stability to extremes of electrode polarization and limited pH range. Frequently polymer formation conditions are incompatible with biomolecules, e.g., requiring nonaqueous solvents or extreme pH. In situ electrochemical formation of conductive polymer/biomolecule composites requires compatibility of the biomolecule with monomers such as pyrrole, aniline and thiophene, which is highly uncertain. If the biosensor is to be utilized in vivo, the matrix must be compatible with the physiological environment. This has not been demonstrated for heterocyclic conductive polymers.
Prior art reports on the encapsulation of biological macromolecules at low temperatures and moderate pH in inorganic sol-gel matrices have been promising, both in regard to preserving bioactivity and substrate/product transport and in imparting superior stability against denaturation (see for example U.S. Pat. No. 5,200,334; B. C. Dave et al., Analytical Chemistry 56, 1130A, 1994). Essentially, the matrix forms a water-permeable conforming shell around the molecules, stabilized by ionic interaction, acid-base complexation and hydrogen bonding. Unfortunately this approach has not been readily transferable to coatings for electrodes since the glasses are electrically insulating. One report of trapping of an enzyme in a semiconductive V.sub.2 O.sub.5 sol-gel glass demonstrated feasibility (V. Glezer and O. Lev, Journal of the American Chemical Society, 115, , see for example 2533, 1993), but the conditions required for formation are generally too aggressive for most biomolecules and V.sub.2 O.sub.5 has poor long-term stability as an electrode in an aqueous environment.
Prior art has also described means for replacing O.sub.2 or other co-factor in oxidoreductase enzymes with an artificial electron acceptor, such as ferricinium U.S. Pat. Nos. 4,545,382, 4,711,245 and 5,378,332. Ferricinium may be generated at the electrode surface by oxidizing ferrocene at &lt;0.2V versus a standard calomel (SCE) reference and will accept an electron from FADH.sub.2. Thus, an electrode containing immobilized ferrocene and GOx held at a potential sufficiently positive to generate ferricinium will provide an amperometric response to glucose, but at a lower potential than if O.sub.2 were used as the electron acceptor. This approach has been used to produce glucose sensors with reduced susceptibility to oxidizable interferents in blood, such as ascorbate. An additional advantage of ferricinium and similar artificial electron acceptors is that electrode response is independent of O.sub.2 tension, which is difficult to control. Some commercial glucose sensors employ glucose oxidase and ferrocene co-immobilized in a carbon matrix. These can only be used once, typically, as the redox mediator will tend to dissolve out of the electrode matrix.
Prior art has also described "wiring" the fast redox couple to the enzyme. Such electrical connection is reviewed by A. Heller (J. Phys. Chem. 92, 3579-3587, 1992). In this approach, the redox couple is covalently immobilized to the matrix or to the protein. For example, matrices comprising polymers with covalently attached redox mediators have been described by Skotheim et al., U.S. Pat. No. 5,264,092. Cross linked polyvinylpyridine complexes of the redox mediator Os(bpy).sub.2 Cl!.sup.+2 are described by Gregg and Heller, Analytical Chemistry 62(3), 1990, pp.258-63. Cited advantages of such electrodes include suppression of leaching of the redox mediators by the surrounding solution (implying reusability).
In view of the success of electron transfer mediators, host matrices are also desired which can also incorporate such mediators or which have electron transfer mediators intrinsic to their structure.
Another feature that is increasingly desired in sensors is their ability to be patterned into arrays using thin film photolithographic processing. Composites based on mixing enzymes with carbon or sol-gel precursors can in principle be printed, but structural size and definition are limited. Processes based on electroforming of conductive polymers onto microfabricated metal electrodes are more favorable, but still have the aforementioned problems of polymer matrix stability.
Hydrous metal oxides have been employed as a thin film pH sensing elements for detecting acidic or basic products of enzyme reactions contained in polymeric overlayers, see for example Tranh-Minh, Biosensors and Bioelectronics 5, 461-471 (1990). These overlayers make for electrodes which require several minutes to reach a full response due to diffusional limitations. They also are not readily amenable to high resolution photolithographic patterning.